Reduction in photosystem II efficiency during a virus-controlled Emiliania huxleyi bloom

During viral infection of Emiliania huxleyi, laboratory studies have shown that photo-system (PS) II efficiency declines during the days post-infection and is thought to be associated with viral-induced interruption of electron transport rates between photosystems. However,measuring the impact of viral infection on PSII function in E. huxleyi populations from natural,taxonomically diverse phytoplankton communities is difficult, and whether this phenomenon occurs in nature is presently unknown. Here, chlorophyll fluorescence analysis was used to assess changes in PSII efficiency throughout an E. huxleyi bloom during a mesocosm experiment off the coast of Norway. Specifically, we aimed to determine whether a measurable suppression of the efficiency of PSII photochemistry could be observed due to viral infection of the natural E. huxleyi populations. During the major infection period prior to bloom collapse, there was a significant reduction in PSII efficiency with an average decrease in maximum PSII photochemical efficiency (Fv/Fm) of 17% and a corresponding 75% increase in maximum PSII effective absorption cross section(σPSII); this was concurrent with a significant decrease in E. huxleyi growth rates and an increase in E. huxleyi virus (EhV) production. As E. huxleyi populations dominated the phytoplankton community and potentially contributed up to 100% of the chlorophyll a pool, we believe that the variable chlorophyll fluorescence signal measured during this period was derived predominantly from E. huxleyi and, thus, reflects changes occurring within E. huxleyi cells. This is the first demonstration of suppression of PSII photochemistry occurring during viral infection of natural coccolithophore populations.

The pathophysiology of eosinophilic esophagitis: Altered mechanisms of antigen detection in the esophagus

Recently, a chronic idiopathic disease of the esophagus has emerged, which is now known as eosinophilic esophagitis (EoE). Incomplete knowledge regarding the pathogenesis of EoE has limited treatment options. EoE is known to be a Th2-type immune-mediated disorder. Based on previous studies in both patients and experimental models, it is possible that an abnormal reaction to antigen mediates the pathophysiology of EoE. In this thesis, symptoms and signs unique to EoE were identified by an age-matched, case-controlled study of 326 patients with EoE and gastroesophageal reflux disease. The molecular mechanisms involved in antigen detection in the esophagus, in relation to EoE were then investigated. Esophageal epithelial cells were found, for the first time, to be capable of acting as non-professional antigen presenting cells, with the ability to engulf, process and present antigen on MHC class II to T helper lymphocytes. Antigen presentation by esophageal epithelial cells was induced by interferon-γ, which is increased in biopsies from patients with EoE. Next, it was discovered that esophageal epithelial cell lines expressed functional toll-like receptor (TLR) 2 and TLR3, but in esophageal mucosal biopsies only infiltrating immune cells (including eosinophils) expressed TLR2 and TLR3. Finally, the potential involvement of IgE in the pathogenesis of esophageal inflammation was investigated. IgE in the esophagus was found to be present on mast cells, which are increased in density in the esophageal mucosae of patients with EoE and especially those with a history of atopy. Mechanisms of antigen detection may mediate the pathophysiology of EoE in the esophagus through antigen presentation by epithelial cells, detection by TLRs on immune cells and detection through IgE on mucosal mast cells. Together, these findings demonstrate that mechanisms of antigen detection may actually contribute to the pathophysiology of EoE. Through increased understanding of the mechanisms of EoE, the results of this thesis may contribute to future therapy.

An Immunohistochemical Study of the Distribution of the Measles Virus Receptors, CD46 and SLAM, In Normal Human Tissues and Subacute Sclerosing Panencephalitis

We have compared the expression of the known measles virus (MV) receptors, membrane cofactor protein (CD46) and the signaling lymphocyte-activation molecule (SLAM), using immunohistochemistry, in a range of normal peripheral tissues (known to be infected by MV) as well as in normal and subacute sclerosing panencephalitis (SSPE) brain. To increase our understanding of how these receptors could be utilized by wild-type or vaccine strains in vivo, the results have been considered with regard to the known route of infection and systemic spread of MV. Strong staining for CD46 was observed in endothelial cells lining blood vessels and in epithelial cells and tissue macrophages in a wide range of peripheral tissues, as well as in Langerhans' and squamous cells in the skin. In lymphoid tissues and blood, subsets of cells were positive for SLAM, in comparison to CD46, which stained all nucleated cell types. Strong CD46 staining was observed on cerebral endothelium throughout the brain and also on ependymal cells lining the ventricles and choroid plexus. Comparatively weaker CD46 staining was observed on subsets of neurons and oligodendrocytes. In SSPE brain sections, the areas distant from lesion sites and negative for MV by immunocytochemistry showed the same distribution for CD46 as in normal brain. However, cells in lesions, positive for MV, were negative for CD46. Normal brain showed no staining for SLAM, and in SSPE brain only subsets of leukocytes in inflammatory infiltrates were positive. None of the cell types most commonly infected by MV show detectable expression of SLAM, whereas CD46 is much more widely expressed and could fulfill a receptor function for some wild-type strains. In the case of wild-type stains, which are unable to use CD46, a further as yet unknown receptor(s) would be necessary to fully explain the pathology of MV infection.

Measles virus superinfection immunity and receptor redistribution in persistently infected NT2 cells

A recombinant measles virus (MV) expressing red fluorescent protein (MVDsRed1) was used to produce a persistently infected cell line (piNT2-MVDsRed1) from human neural precursor (NT2) cells. A similar cell line (piNT2-MVeGFP) was generated using a virus that expresses enhanced green fluorescent protein. Intracytoplasmic inclusions containing the viral nucleocapsid protein were evident in all cells and viral glycoproteins were present at the cell surface. Nevertheless, the cells did not release infectious virus nor did they fuse to generate syncytia. Uninfected NT2 cells express the MV receptor CD46 uniformly over their surface, whereas CD46 was present in cell surface aggregates in the piNT2 cells. There was no decrease in the overall amount of CD46 in piNT2 compared to NT2 cells. Cell-to-cell fusion was observed when piNT2 cells were overlaid onto confluent monolayers of MV receptor-positive cells, indicating that the viral glycoproteins were correctly folded and processed. Infectious virus was released from the underlying cells, indicating that persistence was not due to gross mutations in the virus genome. Persistently infected cells were superinfected with MV or canine distemper virus and cytopathic effects were not observed. However, mumps virus could readily infect the cells, indicating that superinfection immunity is not caused by general soluble antiviral factors. As MVeGFP and MVDsRed1 are antigenically indistinguishable but phenotypically distinct it was possible to use them to measure the degree of superinfection immunity in the absence of any cytopathic effect. Only small numbers of non-fusing green fluorescent piNT2-MVDsRed1 cells (1 : 300 000) were identified in which superinfecting MVeGFP entered, replicated and expressed its genes.

Dynamics of Viral RNA Synthesis during Measles Virus Infection

We propose a reference model of the kinetics of a viral RNA-dependent RNA polymerase (vRdRp) activities and its regulation during infection of eucaryotic cells. After measles virus infects a cell, mRNAs from all genes immediately start to accumulate linearly over the first 5 to 6 h and then exponentially until approximately 24 h. The change from a linear to an exponential accumulation correlates with de novo synthesis of vRdRp from the incoming template. Expression of the virus nucleoprotein (N) prior to infection shifts the balance in favor of replication. Conversely, inhibition of protein synthesis by cycloheximide favors the latter. The in vivo elongation speed of the viral polymerase is approximately 3 nucleotides/s. A similar profile with fivefold-slower kinetics can be obtained using a recombinant virus expressing a structurally altered polymerase. Finally, virions contain only encapsidated genomic, antigenomic, and 5'-end abortive replication fragment RNAs.